CN115287300A - Method for enhancing expression of ExpiCHO cell transient transfection antibody - Google Patents

Abstract

The invention discloses a method for enhancing expression of an antibody transiently transfected by ExpiCHO cells, which comprises the steps of mixing an ExpiCHO cell suspension obtained by the method for transiently transfecting the ExpiCHO cells and then carrying out antibody expression by a method for promoting expression of the antibody after transient transfection of the ExpiCHO cells. Compared with the traditional transfection method, the method can obtain higher expression level of the target protein of the Expicho cells.

Description

A method for enhancing the expression of antibodies for transient transfection in ExpiCHO cells

technical field

The invention relates to the field of biotechnology, in particular to a method for enhancing the expression of antibodies for transient transfection of ExpiCHO cells.

Background technique

Production of recombinant proteins up to milligram or gram scale is an essential process in preclinical, biochemical, biophysical and pharmaceutical research for many research groups and pharmaceutical companies. The production of recombinant proteins in mammalian cell systems has become an expression recombination in the field of biopharmaceuticals because of its ability to optimally express high-molecular-weight protein products, make proteins with complex structures fold correctly, and provide suitable post-translational modifications to glycoproteins. The best choice for protein.

The traditional mammalian cell expression system is to construct a stable cell expression system that integrates foreign genes into the host cell chromosome. Constructing a stable strain expression system to produce recombinant proteins requires long-term screening after DNA is introduced into cells, and then a cell line with high expression and stability can be determined. This process usually takes several months and takes a lot of manpower to complete.

As a mammalian protein expression system, CHO cells (Chinese hamster ovary cell, l, Chinese hamster ovary cells) are widely used to express recombinant DNA proteins. For example, ExpiCHO cells are non-agglomerated and non-aggregated in the matching ExpiCHO expression medium, and can grow to very high cell densities, and can produce higher protein yields through transient transfection. Transient transfection of ExpiCHO cells allows rapid, high-yield transient protein production, allowing laboratories to develop protein biotherapeutics from CHO-expressed proteins to completion during drug development.

With the rapid development of life sciences and biopharmaceuticals, a large number of proteins are used in basic research and drug development, and there is an urgent need for a method that can obtain recombinant proteins in a short time. Transient gene expression came into being. Unlike the stable strain expression system, the exogenous DNA of transient transfection is not integrated into the host cell DNA, and its number in the cell usually decreases with cell division, so protein expression can only be maintained It takes several days to more than ten days, but its advantage is that the gene expression product can be obtained in just a few days, and its versatility is strong. However, compared with the establishment of a stable strain expression system, the production of recombinant proteins by transient transfection has the disadvantages of low transfection efficiency and low expression level, which greatly limits the application of this method. In addition, the commonly used transient transfection method of CHO cells has a low transfection efficiency, and the expression yield of the target gene in the transfected cells is low, which cannot meet the actual needs. Therefore, how to improve the efficiency of transient transfection and increase the expression level has become an urgent problem to be solved in daily operation.

In view of this, the present invention is proposed.

Contents of the invention

The present invention provides a method for enhancing the expression of transiently transfected antibodies in ExpiCHO cells to solve the above-mentioned problems in the prior art.

The scheme of the present invention is:

A method for enhancing the expression of antibodies by transient transfection of ExpiCHO cells. The mixed ExpiCHO cell suspension obtained by the transient transfection method of ExpiCHO cells is then used to express antibodies by promoting the expression of antibodies after transient transfection of ExpiCHO cells.

As a preferred technical solution, the transient transfection method of ExpiCHO cells comprises the following steps:

1) Subculture the ExpiCHO cells one day before the transient transfection, adjust the cell density to 2×10 ⁶ cells/ml, and place a shaker flask containing a certain volume of cells in a shaker;

2) On the day of transient transfection, count the ExpiCHO cells, dilute with fresh medium to adjust the cell density to 4×10 ⁶ cells/ml, and the cell viability is above 95%, to obtain an ExpiCHO cell suspension;

3) For preparation before transfection, mix the prepared liquid A and liquid B and let it stand for a period of time to obtain a mixed drop;

4) Add the mixed drop into the ExpiCHO cell suspension, and cultivate according to the cell culture conditions.

As a preferred technical solution, the liquid A includes plasmids containing antibody light and heavy chains and PBS buffer, and the ratio of antibody light chain-containing plasmids to antibody heavy chain-containing plasmids in the antibody light and heavy chain-containing plasmids is 1:1. Then the plasmid containing the light and heavy chains of the antibody was mixed with 600 μl of PBS buffer to obtain liquid A; the liquid B included PEI transfection reagent and PBS buffer; The cell number ratio of the ExpiCHO cell suspension is 20 μg: 4×10 ⁷ cells-12×10 ⁷ cells; the mass ratio of the total plasmid amount of the antibody light and heavy chain plasmids to the added amount of the transfection reagent is 1 :2～1:6.

As a preferred technical solution, the ExpiCHO cells should be passaged once within 24 hours of transfection; the shake flask is a round-bottom shaker flask with a size of 250ml to 1000ml for passaging, and a round-bottom shaker flask with a size of 125ml for transfection. bottle; the certain volume in the step 1) is one of 10ml, 20ml, and 30ml; the method of standing in the step 3) is the condition that the room temperature is not lower than 29°C, and the standing time is 10 to 20 minutes or The resting is one of the standing water baths at 29° C.; the cell subculture and cell culture conditions are 37° C., 8% CO 2 , and the speed of the shaker is 110 rpm.

As a preferred technical scheme, the ratio of the total plasmid quantity of the antibody light and heavy chain to the cell number of the ExpiCHO cell suspension is 20 μg: ⁸ ×107 cells; the total plasmid quantity of the antibody light and heavy chain plasmid is the same as the The mass ratio of the amount of transfection reagent added was 1:4.

As a preferred technical solution, the ExpiCHO cell suspension also includes a serum-free medium, the serum-free medium is Transpro CD01, and the ExpiCHO cell suspension includes 4mmol/L-6mmol/L of glutamine added before transfection amides.

As a preferred technical solution, the method for promoting antibody expression after transient transfection of ExpiCHO cells comprises the following steps:

Recombinant ExpiCHO cells were obtained after transient transfection, and the temperature was lowered for 4-10 hours after transfection. Hydrolyzate was added 12-24 hours after transfection. The amount of hydrolyzate added was 3g/L-8g/L to promote transfection Cellular protein expression.

As a preferred technical solution, the hydrolyzate is a plant protein hydrolyzate, and the plant protein hydrolyzate includes Bacto malt powder, BBL plant protein hydrolyzate, Difco selected soytone, Difco ultra-filtered yeast hydrolyzate, and BBL yeast extract , Bacto yeast extract, Bacto yeast extract, Oxoid No. 1 plant peptone, Oxoid non-GMO soybean peptone, soybean SE50MK-NK, soybean SE50MK-NK, soybean WGE80M-UF ultrafiltration type, Kerry Hypep-1510, KerryHypep-4601N, A mixture of one or more of Kerry Hypep-YE and Oxoid No. 1 plant peptone.

As a preferred technical solution, at 18 hours after the transfection, 3g/L-8g/L plant protein hydrolyzate is added; the cooling culture condition after the cell transfection is 32°C, 8% CO2, shaking table speed It is 110rpm; the plant protein hydrolyzate is the mixture of Bacto yeast extract, Kerry Hypep-1510 and Difco ultrafiltration yeast hydrolyzate, wherein Bacto yeast extract 5g/L, Kerry Hypep-1510 3g/L, Difco ultrafiltration yeast Hydrolyzate 6g/L.

Application of a transient transfection method of ExpiCHO cells in recombinant protein expression.

Due to the adoption of the above-mentioned technical scheme, a method for enhancing the expression of antibodies by transient transfection of ExpiCHO cells, the mixed ExpiCHO cell suspension obtained by the transient transfection method of ExpiCHO cells is then used to promote the expression of antibodies after transient transfection of ExpiCHO cells. Express.

Advantages of the present invention:

The invention provides a method for promoting the antibody expression of Expicho cells after transient transfection, and promotes the antibody expression of the transfected cells through technological parameters such as cooling culture after transfection and adding a certain amount of hydrolyzate at a certain time after transfection. Compared with the traditional transfection method, a higher expression level of the target protein in Expicho cells can be obtained by using the above method.

Description of drawings

Figure 1 is a photomicrograph of the state of Expicho cells after subculture.

Detailed ways

In order to make up for the above deficiencies, the present invention provides a method for enhancing the expression of transiently transfected antibodies in ExpiCHO cells to solve the above problems in the background technology.

A method for enhancing the expression of antibodies by transient transfection of ExpiCHO cells. The mixed ExpiCHO cell suspension obtained by the transient transfection method of ExpiCHO cells is then used to express antibodies by promoting the expression of antibodies after transient transfection of ExpiCHO cells.

The transient transfection method for ExpiCHO cells involves the following steps:

1) Subculture the ExpiCHO cells one day before the transient transfection, adjust the cell density to 2×10 ⁶ cells/ml, and place a shaker flask containing a certain volume of cells in a shaker;

2) On the day of transient transfection, count the ExpiCHO cells, dilute with fresh medium to adjust the cell density to 4×10 ⁶ cells/ml, and the cell viability is above 95%, to obtain an ExpiCHO cell suspension;

3) For preparation before transfection, mix the prepared liquid A and liquid B and let it stand for a period of time to obtain a mixed drop;

4) Add the mixed drop into the ExpiCHO cell suspension, and cultivate according to the cell culture conditions.

Said liquid A includes plasmids containing light and heavy chains of antibodies and PBS buffer, said plasmids containing light and heavy chains of antibodies includes the ratio of plasmids containing light chains of antibodies to plasmids containing heavy chains of antibodies is 1:1, and then said antibody-containing plasmids The plasmids of the light and heavy chains were mixed with 600 μl of PBS buffer to obtain liquid A; the liquid B included PEI transfection reagent and PBS buffer; the total amount of plasmids of the antibody light and heavy chains and the cells of the ExpiCHO cell suspension The quantity ratio is 20 μg: 4×10 ⁷ cells-12×10 ⁷ cells; the mass ratio of the total plasmid quantity of the antibody light and heavy chain plasmids to the added amount of the transfection reagent is 1:2～1:6 .

The ExpiCHO cells should be subcultured once within 24 hours of transfection; the shake flask is a round bottom shake flask with a specification of 250ml to 1000ml for passage, and a round bottom shake flask with a specification of 125ml for transfection; the step 1 ) in a certain volume of 10ml, 20ml, 30ml; the method of standing still in the step 3) is the condition that the room temperature is not lower than 29°C, and the standing time is 10 to 20 minutes or the standing time is 29 The conditions of cell subculture and cell culture are 37° C., 8% CO 2 , and the speed of the shaker is 110 rpm.

The total plasmid amount of the antibody light and heavy chains and the cell number ratio of the ExpiCHO cell suspension are 20 μg: ⁸ ×107 cells; the total plasmid amount of the antibody light and heavy chain plasmids and the added amount of the transfection reagent The mass ratio is 1:4.

The ExpiCHO cell suspension also includes a serum-free medium, the serum-free medium is TransproCD01, and the ExpiCHO cell suspension includes 4 mmol/L-6 mmol/L of glutamine added before transfection.

The method for promoting antibody expression after transient transfection of ExpiCHO cells comprises the following steps:

Recombinant ExpiCHO cells were obtained after transient transfection, and the temperature was lowered for 4-10 hours after transfection. Hydrolyzate was added 12-24 hours after transfection. The amount of hydrolyzate added was 3g/L-8g/L to promote transfection Cellular protein expression.

The hydrolyzate is a plant protein hydrolyzate, and the plant protein hydrolyzate includes Bacto malt powder, BBL plant protein hydrolyzate, Difco selected soytone, Difco ultrafiltration yeast hydrolyzate, BBL yeast extract, Bacto yeast extract, Bacto yeast extract, Oxoid No. 1 plant peptone, Oxoid non-GMO soybean peptone, soybean SE50MK-NK, soybean SE50MK-NK, soybean WGE80M-UF ultrafiltration type, Kerry Hypep-1510, Kerry Hypep-4601N, KerryHypep-YE and Oxoid A mixture of one or more of No. 1 plant peptones.

At 18 hours after the transfection, 3g/L-8g/L plant protein hydrolyzate was added; the cooling culture conditions after cell transfection were 32°C, 8% CO2, and the shaker speed was 110rpm; the plant The protein hydrolyzate is a mixture of Bacto yeast extract, Kerry Hypep-1510 and Difco ultra-filtered yeast hydrolyzate, in which Bacto yeast extract is 5g/L, KerryHypep-1510 is 3g/L, and Difco ultra-filtered yeast hydrolyzate is 6g/L.

Application of a transient transfection method of ExpiCHO cells in recombinant protein expression.

The transient transfection of ExpiCHO cells and the method for promoting antibody expression after transfection according to the embodiment,

The general method is based on the expicho cell suspension in the logarithmic growth phase of Transpro CD01 culture medium, and through the method of single factor analysis of the control variable, from the cell number and plasmid ratio, plasmid and transfection at the time of transfection Reagent ratio, cooling time after transfection, time of adding substances after transfection, type and concentration of added substances after transfection, etc. were tested, analyzed the results, and obtained transient transfection process parameters; the following is a step-by-step explanation of the general method.

Taking the mass ratio of the total amount of cells of the Expicho cell suspension in the logarithmic growth phase and the total antibody plasmid as a variable design experiment, adding the transfection reagent and the plasmid to the Expicho cell suspension for transient transfection, analysis As a result, transient transfection process parameters are obtained;

Design an experiment with the amount of transfection reagent added and the mass ratio of the total antibody plasmid as variables, add the transfection reagent and the plasmid to the Expicho cell suspension for transient transfection, analyze the results, and obtain a transient transfection process parameter;

The Expicho recombinant cells obtained by the above transfection method were subjected to cooling culture conditions at different time points after transfection, and the supernatant was collected by centrifugation to detect the expression level of antibody IgG, and the results were analyzed to obtain the process parameters for transient transfection to promote antibody expression;

For the Expicho recombinant cells obtained by the above transfection method, the culture conditions of plant protein hydrolyzate were added at different time points after transfection, the supernatant was collected by centrifugation to detect the expression level of antibody IgG, and the results were analyzed to obtain the results of transient transfection promoting antibody expression. Process parameters;

For the Expicho recombinant cells obtained by the above transfection method, 18 hours after transfection, the culture conditions of adding 3g/L～8g/L plant protein hydrolyzate were added, the supernatant was collected by centrifugation to detect the expression of antibody IgG, and the results were analyzed to obtain instantaneous Process parameters for transfection to facilitate antibody expression.

Transpro CD01 is a universal transient medium specially designed and developed by Duoning Bio for HEK293 cells and CHO cells. This product can be used for subculture, high-density culture and transient transfection culture of cells at the same time. During the transient transfection No centrifugation is required. Transpro CD01 is suitable for HEK293 series cells such as HEK 293, Expi293F, 293F, 293E and CHO series cells such as expiCHOS and CHOS for transient transfection and expression culture of antibodies, recombinant proteins and viruses during the research and development process. The product is a completely chemically defined medium, no animal-derived ingredients, no protein ingredients, no animal or plant-derived protein hydrolyzates, and no growth factors. This product does not contain HT and anti-caking agents. Transpro CD01 liquid packaging does not contain L-glutamine, and an additional 4-6mM L-glutamine is required for use.

The cell density of ExpiCHO cell suspension is 4×10 ⁷ cells to 12× ¹⁰⁷ cells: 20 μg total amount of plasmid (light chain:heavy chain = 1:1), the mass ratio of total plasmid amount to the added amount of transfection reagent is 1:2-1:6.

The transfected Expicho cell suspension was cultured at 5 hours, 8 hours or 10 hours after transfection at a time point of cooling.

Plant hydrolyzates were added to the transfected Expicho cell suspension at 12, 18 or 24 hours post-transfection time points.

Add different types and concentrations of plant hydrolyzate after transfection; it is worth noting that the quality and concentration of the hydrolyzate from different manufacturers vary greatly.

Using Transpro CD01 for a subculture, and using fresh Transpro CD01 to directly adjust the cell density without centrifugation can greatly preserve the cell viability and cell state, which is also a key step in the present invention.

The Expicho cell suspension obtained by direct centrifugation without subculture was used, and the specific operation method is summarized as follows:

(1) In this experiment, the well-growing Expicho cells were taken, and the cell viability was above 95%. The cells were collected by centrifugation at room temperature and 800rmp for 5 minutes, and the cell density was adjusted to ^4X106 cells/ml with fresh Transpro CD01 medium, and the final concentration was added 4-6mmol/L glutamine.

(2) Add the adjusted Expicho cell suspension into the 125ml Donin shake flask according to the total number of cells 4X10 ⁷ cells to 12X10 ⁷ cells (i.e. 10ml~30ml cell suspension), and put the treated Expicho cell shake flask temporarily Placed in a shaker for culture, the culture parameter temperature is 37° C., the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

(3) Prepare the transfection system during this period, and the preparation method is as follows:

①The quality of the antibody light chain plasmid and the antibody heavy chain plasmid is 10ug each to form a 20ug antibody plasmid mixture;

② Preparation of solution A: Add 600 μl of PBS to the total plasmid tube.

③Preparation of solution B: Take 40μg, 80μg, and 120μg of PEI, and add them to 600μl of PBS to equilibrate.

④ Place the above liquids A and B in room temperature at 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

⑤ Slowly drop the well-balanced solution A into solution B, and continue to place the AB mixed solution at a room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

(4) The well-balanced AB solution was slowly dripped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

(5) 5-10 hours after the transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32°C, the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

(6) Add a certain concentration of hydrolyzate to the culture system within 24 hours after transfection.

(7) Sampling was taken every day to detect cell growth. When the cell viability was lower than 95%, 1 ml of cells was taken, centrifuged at 4° C. and 10,000 rpm for 10 minutes, and the supernatant was collected, and the antibody titer in the supernatant was detected by liquid phase.

In one of the examples, all the optimal methods after optimization were used, the results were analyzed, and the optimal process parameters for transient transfection to promote antibody expression were obtained. The specific operation methods are summarized as follows:

(1) Take well-growing Expicho cells, subculture the cells 24 hours before transfection, and adjust the cell density to 2X106 cells/ml with Transpro CD01 medium supplemented with 4-6mmol/L glutamine. The survival rate is over 95%;

(2) The above-mentioned Expicho cell suspension was placed in a 1000ml Donin shake flask and cultured in a shaker. The temperature of the shaker was set at 37° C., the concentration of carbon dioxide was 8%, and the speed of the shaker was set at 110 rpm.

(3) After 24 hours, take the Expicho cell suspension for counting and state observation. The cell viability is above 95%. The cell density is adjusted to ⁴ ×10 cells/ml with fresh Transpro CD01 medium, and a final concentration of 4- 6mmol/L glutamine.

(4) Add the adjusted density of Expicho cell suspension into 125ml Donin shake flasks according to the total number of cells of 4X10 ⁷ cells to 12X10 ⁷ cells into volumes of 10ml, 20ml, and 30ml respectively, and shake the treated Expicho cells Temporarily placed in a shaker for culture, the culture parameter temperature is 37° C., the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

(5) Prepare the transfection system during this period, and the preparation method is as follows:

①The quality of the antibody light chain plasmid and the antibody heavy chain plasmid is 10ug each to form a 20ug antibody plasmid mixture;

② Preparation of solution A: Add 600 μl of PBS to the total plasmid tube.

③Preparation of solution B: Take 40μg, 80μg, and 120μg of PEI, respectively, and add them into 600μl of PBS to balance.

④ Place the above liquids A and B in room temperature at 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

⑤ Slowly drop the well-balanced solution A into solution B, and continue to place the AB mixed solution at a room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

(6) The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

(7) At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

(8) 18 hours after transfection, 5 g/L of Bacto yeast extract, 3 g/L of KerryHypep-1510, and 6 g/L of Difco ultrafiltered yeast hydrolyzate were added to the culture system.

(9) Take samples every day to detect cell growth. When the cell viability is lower than 95%, take 1ml of cells, centrifuge at 4°C and 10,000rmp for 10 minutes, collect the supernatant, and detect the antibody titer in the supernatant by liquid phase.

The transfection method before the original optimization was used, and the specific operation method is summarized as follows:

(1) Take well-growing Expicho cells, the cell viability is above 95%, adjust the cell density to ^4X106 cells/ml with fresh Dynamis medium, add 30ml of culture volume to 125ml Donin shake flask, and place temporarily Cultivate in a shaker, the culture parameter temperature is 37° C., the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

(2) Prepare the transfection system during this period, and the preparation method is as follows:

①The quality of the antibody light chain plasmid and the antibody heavy chain plasmid is 10ug each to form a 20ug antibody plasmid mixture;

② Preparation of solution A: Add 600 μl of PBS to the total plasmid tube.

③Preparation of solution B: Take 40 μg of PEI and add to 600 μl of PBS to equilibrate.

④ Place the above liquids A and B at room temperature and let them stand for 10 minutes to balance.

⑤ Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature for 20 minutes to balance.

(3) The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

(4) Take a sample every day to check the cell growth. When the cell viability is lower than 95%, take 1ml of cells, centrifuge at 4°C and 10000rmp for 10 minutes, collect the supernatant, and detect the antibody titer in the supernatant by liquid phase.

In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

The following is the specific embodiment part:

The reagents and instruments used in the examples are all conventional choices in the art unless otherwise specified. The experimental methods without specific conditions indicated in the examples are usually implemented according to conventional conditions, such as the conditions described in literature, books or the method recommended by the kit manufacturer. The reagents used in the examples are all commercially available.

Unless otherwise specified, in the following examples, the antibody heavy and light chain plasmids contain the expression plasmids respectively containing the antibody heavy chain and the antibody light chain, which were purchased from Excellgene. The hydrolyzate was purchased from BD Company and KERRY Company respectively.

Example 1:

The total number of Expicho cells, the total amount of antibody plasmids, the ratio of the amount of transfection reagent PEI and the expression of antibodies,

In this example, the well-growing Expicho cells were taken, and the cells were subcultured 24 hours before transfection, and the cell density was adjusted to 2X106 cells/ml with Transpro ^CD01 medium supplemented with 4-6mmol/L glutamine , the cell viability is above 95%;

The above-mentioned Expicho cell suspension was placed in a 250ml-500ml Donin shake flask and cultured in a shaker. The temperature of the shaker was set at 37° C., the concentration of carbon dioxide was 8%, and the shaker speed was set at 110 rpm.

After 24 hours, take the Expicho cell suspension for counting and state observation. The cell viability is above 95%. Use fresh Transpro CD01 medium to adjust the cell density to ^4X106 cells/ml, and add a final concentration of 4-6mmol/L glutamine.

The adjusted density of the Expicho cell suspension is converted into volumes of 10ml, 20ml, and 30ml according to the total number of cells from ^4X107 cells to ^12X107 cells, and added to 125ml Donin shake flasks, and the treated Expicho cell shake flasks are temporarily placed in Cultivate in a shaker, the culture parameter temperature is 37° C., the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 40 μg, 80 μg, and 120 μg of PEI respectively, and add them to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

Samples were taken every day to detect cell growth. When the cell viability was lower than 95%, 1ml of cells was taken, centrifuged at 4°C and 10,000rmp for 10 minutes, and the supernatant was collected, and the antibody titer in the supernatant was detected by liquid phase.

Figure 1 shows that the subcultured cells are in the logarithmic growth phase, and the cells are in good condition and suitable for transfection.

Table 2 shows the total number of Expicho cells: antibody plasmid mass = 8X10 ⁷ cells (ie 20ml cell volume): 20 μg, the antibody expression level is the highest; transfection reagent PEI mass: antibody plasmid mass = 4:1, the antibody titer is the highest.

Table 2

Therefore, in the following examples, the total number of Expicho cells is preferably ^8X107 cells (i.e. 20ml cell volume), the transfection conditions of 80 μg of transfection reagent mass and 20 μg of antibody plasmid total amount are the experimental basis for subsequent enhanced antibody expression after transfection .

Example 2:

Cooling time points and antibody expression after transfection

In this example, the well-growing Expicho cells were taken, and the cells were subcultured 24 hours before transfection, and the cell density was adjusted to 2X106 cells/ml with Transpro ^CD01 medium supplemented with 4-6mmol/L glutamine , the cell viability is above 95%;

The above-mentioned Expicho cell suspension was placed in a 250ml-500ml donin shake flask and cultured in a shaker. The temperature of the shaker was set at 37° C., the concentration of carbon dioxide was 8%, and the shaker speed was set at 110 rpm.

After 24 hours, take the Expicho cell suspension for counting and state observation. The cell viability is above 95%. Use fresh Transpro CD01 medium to adjust the cell density to ^4X106 cells/ml, and add a final concentration of 4-6mmol/L glutamine.

Add the adjusted Expicho cell suspension into a 125ml Donin shake flask according to the total number of cells ^8X107 cells (20ml), and temporarily place the treated Expicho cell shake flask in a shaker for culture, and the culture parameter temperature is 37°C , the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 80 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dripped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 5 hours, 8 hours or 10 hours after the transfection, the shake flasks were cultured in a cooling culture condition, the culture parameters were 32° C., 8% carbon dioxide concentration, and the shaker speed was set at 110 rpm.

Samples were taken every day to detect cell growth. When the cell viability was lower than 95%, 1ml of cells was taken, centrifuged at 4°C and 10,000rmp for 10 minutes, and the supernatant was collected, and the antibody titer in the supernatant was detected by liquid phase.

The results shown in Table 3 are the relationship between cooling conditions and antibody expression. It can be seen that cooling 8 hours after transfection is most suitable for antibody expression.

table 3

Therefore, the subsequent hydrolyzate addition time point is carried out on this basis.

Example 3:

Post-transfection hydrolyzate addition time points and antibody expression

In this example, the well-growing Expicho cells were taken, and the cells were subcultured 24 hours before transfection, and the cell density was adjusted to 2X106 cells/ml with Transpro ^CD01 medium supplemented with 4-6mmol/L glutamine , the cell viability is above 95%;

The above-mentioned Expicho cell suspension was placed in a 250ml-500ml donin shake flask and cultured in a shaker. The temperature of the shaker was set at 37° C., the concentration of carbon dioxide was 8%, and the shaker speed was set at 110 rpm.

After 24 hours, take the Expicho cell suspension for counting and state observation. The cell viability is above 95%. Use fresh Transpro CD01 medium to adjust the cell density to ^4X106 cells/ml, and add a final concentration of 4-6mmol/L glutamine.

Add the adjusted density of the Expicho cell suspension into a 125ml Donin shake flask according to the total number of cells ^8X107 cells (20ml), and temporarily place the treated Expicho cell shake flask in a shaker for culture, and the culture parameter temperature is 37°C , the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

Prepare the transfection system during this period, and the preparation method is as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Preparation of solution B: Take 80 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced solution A into solution B, and continue to place the AB mixed solution at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dripped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 12 hours, 18 hours or 24 hours after transfection, 3 g/L of Kerry Hypep-1510 hydrolyzate (this hydrolyzate is a plant hydrolyzate known to help the growth of CHO cells) was added to the culture system.

Samples were taken every day to detect cell growth. When the cell viability was lower than 95%, 1ml of cells was taken, centrifuged at 4°C and 10,000rmp for 10 minutes, and the supernatant was collected, and the antibody titer in the supernatant was detected by liquid phase.

The results shown in Table 4 are the relationship between the addition time of the hydrolyzate and the expression of the antibody. It can be seen that adding the plant hydrolyzate 18 hours after transfection is most suitable for the expression of the antibody.

Table 4

Therefore, the type and content of the subsequent hydrolyzate addition is carried out on this basis.

Example 4:

The type and amount of hydrolyzate and antibody expression after transfection

In this example, the well-growing Expicho cells were taken, and the cells were subcultured 24 hours before transfection, and the cell density was adjusted to 2X106 cells/ml with Transpro ^CD01 medium supplemented with 4-6mmol/L glutamine , the cell viability is above 95%;

The above-mentioned Expicho cell suspension was placed in a 250ml-500ml Donin shake flask and cultured in a shaker. The temperature of the shaker was set at 37° C., the concentration of carbon dioxide was 8%, and the shaker speed was set at 110 rpm.

After 24 hours, take the Expicho cell suspension for counting and state observation. The cell viability is above 95%. Use fresh Transpro CD01 medium to adjust the cell density to ^4X106 cells/ml, and add a final concentration of 4-6mmol/L glutamine.

Add the adjusted density of the Expicho cell suspension into a 125ml Donin shake flask according to the total number of cells ^8X107 cells (20ml), and temporarily place the treated Expicho cell shake flask in a shaker for culture, and the culture parameter temperature is 37°C , the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 80 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

3 g/L plant hydrolyzate was added to the culture system 18 hours after transfection.

The vegetable protein hydrolyzate that embodiment adds is as shown in the following table:

control none Experiment 1 Bacto malt powder Experiment 2 BBL vegetable protein hydrolyzate Experiment 3 Difco Select Soytone Experiment 4 Difco Ultrafiltration Yeast Hydrolyzate Experiment 5 BBL yeast extract Experiment 6 Bacto Yeast Extract Experiment 7 Oxoid Yeast Extract Experiment 8 Oxoid One Plant Peptone Experiment 9 Oxoid Non-GMO Soy Peptone Experiment 10 Soybean SE50MK-NK Experiment 11 Soybean SE70M-UF ultrafiltration type Experiment 12 Soybean WGE80M-UF ultrafiltration type Experiment 13 Kerry Hypep-1510 Experiment 14 Kerry Hypep-4601N Experiment 15 Kerry Hypep-YE Experiment 16 Oxoid One Plant Peptone

5 g/L plant hydrolyzate was added to the culture system 18 hours after transfection.

The vegetable protein hydrolyzate that embodiment adds is as shown in the following table:

Eighteen hours after transfection, 8 g/L plant hydrolyzate was added to the culture system. The vegetable protein hydrolyzate that embodiment adds is as shown in the following table:

control none Experiment 1 Bacto malt powder Experiment 2 BBL vegetable protein hydrolyzate Experiment 3 Difco Select Soytone Experiment 4 Difco Ultrafiltration Yeast Hydrolyzate Experiment 5 BBL yeast extract Experiment 6 Bacto Yeast Extract Experiment 7 Oxoid Yeast Extract Experiment 8 Oxoid One Plant Peptone Experiment 9 Oxoid Non-GMO Soy Peptone Experiment 10 Soybean SE50MK-NK Experiment 11 Soybean SE70M-UF ultrafiltration type Experiment 12 Soybean WGE80M-UF ultrafiltration type Experiment 13 Kerry Hypep-1510 Experiment 14 Kerry Hypep-4601N Experiment 15 Kerry Hypep-YE Experiment 16 Oxoid One Plant Peptone

Samples were taken every day to detect cell growth. When the cell viability was lower than 95%, 1ml of cells was taken, centrifuged at 4°C and 10,000rmp for 10 minutes, and the supernatant was collected, and the antibody titer in the supernatant was detected by liquid phase.

When 5g/L of Bacto yeast extract, 3g/L of Kerry Hypep-15103g/L, and 6g/L of Difco ultrafiltered yeast hydrolyzate were added to the transfected culture system shown in Table 5, the antibody expression of Expicho cells was the highest.

table 5

Example 5:

The optimized method for transfection and antibody expression was compared with the transfection method and antibody expression before optimization. This example is divided into the comparison of antibody expression before and after optimization. The methods before and after optimization are introduced below.

Optimized methods for transfection and improved antibody expression:

Take well-growing Expicho cells, subculture the cells 24 hours before transfection, and adjust the cell density to 2X106 cells/ml with Transpro CD01 medium supplemented with 4-6mmol/L glutamine, and the cell viability is at above 95;

The above-mentioned Expicho cell suspension was placed in a 1000ml Donin shake flask and cultured in a shaker. The temperature of the shaker was set at 37°C, the concentration of carbon dioxide was 8%, and the speed of the shaker was set at 110rpm.

After 24 hours, take the Expicho cell suspension for counting and state observation. The cell viability is above 95%. Use fresh Transpro CD01 medium to adjust the cell density to ^4X106 cells/ml, and add a final concentration of 4-6mmol/L glutamine.

The Expicho cell suspension with adjusted density is added in the 125ml Donin shake flask according to the total number of cells 8× ^{10 7} cells (i.e. the volume is 20ml), and the processed Expicho cell shake flask is temporarily placed in a shaker for cultivation, and the culture parameter temperature is 37°C, carbon dioxide concentration 8%, shaker speed set at 110rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 80 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at a room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 18 hours after transfection, 5 g/L of Bacto yeast extract, 3 g/L of Kerry Hypep-1510, and 6 g/L of Difco ultrafiltered yeast hydrolyzate were added to the culture system.

Transfection method before optimization:

Take the well-growing Expicho cells, the cell viability is above 95%, adjust the cell density to ^4X106 cells/ml with fresh Dynamis medium, add 30ml of culture volume to a 125ml Donin shaker flask, and temporarily place it in a shaker For cultivation, the cultivation parameter temperature is 37° C., the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 40 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above liquids A and B at room temperature for 10 minutes to balance.

The well-balanced solution A was slowly dropped into solution B, and the AB mixture was left at room temperature for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

The detection methods of the above two methods are the same. Samples are taken every day to detect the growth of cells. When the cell viability is lower than 95%, take 1ml of cells, centrifuge at 4°C and 10,000rmp for 10 minutes, collect the supernatant, and detect the concentration in the supernatant by liquid phase. Antibody titer.

Table 6 shows that the optimized transfection and the method of promoting antibody expression after transfection are significantly better than the transfection method before optimization.

Table 6

Embodiment 6:

In this example, the expression of antibodies under optimized conditions at different stages is compared, and the specific implementation methods are as follows:

Experiment 1: Ratio of the total number of cells to the total mass of antibody plasmids

In this experiment, the well-growing Expicho cells were taken, the cell viability was above 95%, and the cells were collected by centrifugation at room temperature and 800rmp for 5 minutes, and the cell density was adjusted to ^4X106 cells/ml with fresh Transpro CD01 medium, and the final concentration was added to 4-6mmol /L Glutamine.

The adjusted density of the Expicho cell suspension is converted into volumes of 10ml, 20ml, and 30ml according to the total number of cells from ^4X107 cells to ^12X107 cells, and added to 125ml Donin shake flasks, and the treated Expicho cell shake flasks are temporarily placed in Cultivate in a shaker, the culture parameter temperature is 37° C., the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 40 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

Add Kerry Hypep-1510 3g/L to the culture system 18 hours after transfection.

Experiment 2: Total Mass Ratio of Transfection Reagent and Antibody Plasmid

In this experiment, the well-growing Expicho cells were taken, the cell viability was above 95%, and the cells were collected by centrifugation at room temperature and 800rmp for 5 minutes, and the cell density was adjusted to ^4X106 cells/ml with fresh Transpro CD01 medium, and the final concentration was added to 4-6mmol /L Glutamine.

Add the Expicho cell suspension with adjusted density into 125ml Donin shake flask according to the total number of cells 8X10 ⁷ cells (i.e. 20ml cell suspension), and temporarily place the treated Expicho cell shake flask in a shaker for cultivation. The temperature is 37°C, the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Preparation of solution B: Take 40 μg, 80 μg, and 120 μg of PEI, and add them to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

Add Kerry Hypep-1510 3g/L to the culture system 18 hours after transfection.

Experiment 3: Cooling time after transfection

In this experiment, the well-growing Expicho cells were taken, the cell viability was above 95%, and the cells were collected by centrifugation at room temperature and 800rmp for 5 minutes, and the cell density was adjusted to ^4X106 cells/ml with fresh Transpro CD01 medium, and the final concentration was added to 4-6mmol /L Glutamine.

Add the Expicho cell suspension with adjusted density into 125ml Donin shake flask according to the total number of cells 8X10 ⁷ cells (i.e. 20ml cell suspension), and temporarily place the treated Expicho cell shake flask in a shaker for cultivation. The temperature is 37°C, the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 80 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 5 hours, 8 hours or 10 hours after the transfection, the shake flasks were cultured in a cooling culture condition, the culture parameters were 32° C., 8% carbon dioxide concentration, and the shaker speed was set at 110 rpm.

Add Kerry Hypep-1510 3g/L to the culture system 18 hours after transfection.

Experiment 4: Time of hydrolyzate addition after transfection

In this experiment, the well-growing Expicho cells were taken, the cell viability was above 95%, and the cells were collected by centrifugation at room temperature and 800rmp for 5 minutes, and the cell density was adjusted to ^4X106 cells/ml with fresh Transpro CD01 medium, and the final concentration was added to 4-6mmol /L Glutamine.

Add the Expicho cell suspension with adjusted density into 125ml Donin shake flask according to the total number of cells 8X10 ⁷ cells (i.e. 20ml cell suspension), and temporarily place the treated Expicho cell shake flask in a shaker for cultivation. The temperature is 37°C, the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 80 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

Add Kerry Hypep-1510 3g/L to the culture system 12 hours, 18 hours or 24 hours after transfection.

Experiment 5: The type and amount of hydrolyzate added after transfection

In this experiment, the well-growing Expicho cells were taken, the cell viability was above 95%, and the cells were collected by centrifugation at room temperature and 800rmp for 5 minutes, and the cell density was adjusted to ^4X106 cells/ml with fresh Transpro CD01 medium, and the final concentration was added to 4-6mmol /L Glutamine.

Add the Expicho cell suspension with adjusted density into 125ml Donin shake flask according to the total number of cells ^8X107 cells (ie 20ml cell suspension), and temporarily place the treated Expicho cell shake flask in a shaker for cultivation, and the culture parameter temperature The temperature is 37°C, the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 80 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above-mentioned liquids A and B at a room temperature of 29°C or in preheated warm water at 29°C, and let them stand for 15 minutes to balance.

Slowly drop the well-balanced liquid A into liquid B, and continue to place the AB mixture at room temperature of 29°C or in preheated warm water at 29°C, and let it stand for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

At 8 hours after transfection, the shake flask was cultured in a cooling culture condition, the culture parameter temperature was 32° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

The following types and concentrations of hydrolyzate were added to the culture system 18 hours after transfection: 500 μl PBS, Kerry Hypep-1510 3 g/L, Difco ultrafiltered yeast hydrolyzate 3 g/L.

It should be noted that in Experiment 1-Experiment 5, in order to make the experiment more convincing, the transfected cells in the early stage were not subcultured in Examples 1-5, but were directly centrifuged from Expicho cell seeds, which can To keep the cell state consistent, when studying the influence of a single variable on the expression of antibodies after transfection in Experiment 1-Experiment 5, only the variable factor was changed in each experiment, and other conditions were the optimal conditions optimized in Examples 1-5.

Control group: traditional Dynamis transient method

Take the well-growing Expicho cells, the cell viability is above 95%, adjust the cell density to ^4X106 cells/ml with fresh Dynamis medium, add 30ml of culture volume to a 125ml Donin shaker flask, and temporarily place it in a shaker For cultivation, the cultivation parameter temperature is 37° C., the carbon dioxide concentration is 8%, and the shaker speed is set at 110 rpm.

During this period, the transfection system was prepared, and the preparation method was as follows:

Containing 10ug of antibody light chain plasmid and antibody heavy chain plasmid each, to form a 20ug antibody plasmid mixture;

Prepare Solution A: Add 600 μl of PBS to the total plasmid tube.

Prepare solution B: Take 40 μg of PEI and add to 600 μl of PBS to equilibrate.

Place the above liquids A and B at room temperature for 10 minutes to balance.

The well-balanced solution A was slowly dropped into solution B, and the AB mixture was left at room temperature for 20 minutes to balance.

The well-balanced AB solution was slowly dropped into the above-mentioned Expicho cell suspension to be transfected, and placed in a shaker for culture. The culture parameter temperature was 37° C., the carbon dioxide concentration was 8%, and the shaker speed was set at 110 rpm.

The detection methods of the above experiments and control experiments are the same. Samples are taken every day to detect cell growth. When the cell viability is lower than 95%, take 1ml of cells, centrifuge at 4°C and 10,000rmp for 10 minutes, collect the supernatant, and detect the supernatant by liquid phase. Antibody titers in .

The results are shown in Table 7. Experiment 2: The total mass ratio of transfection reagent and antibody plasmid, Experiment 3: Cooling time after transfection and Experiment 5: The contribution of the type and amount of hydrolyzate added to the antibody expression after transfection The value is the highest, followed by experiment 1: the total number of cells and the total mass ratio of antibody plasmids, and experiment 4: the time of hydrolyzate addition after transfection.

Table 7

In summary, the above-mentioned transient transfection method of ExpiCHO cells can significantly increase the antibody expression after antibody plasmid transfection into ExpiCHO cell suspension, which provides a reference for the transient transfection of ExpiCHO cells to express antibodies.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Variations and improvements are possible, which fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. a method for enhancing ExpiCHO cell transient transfection antibody expression, is characterized in that: the mixed ExpiCHO cell suspension obtained by the transient transfection method of ExpiCHO cell then carries out antibody by the method for promoting the expression of antibody after the transient transfection of ExpiCHO cell Express. 2. the method for enhancing ExpiCHO cell transient transfection antibody expression as described in right 1, is characterized in that, the transient transfection method of ExpiCHO cell comprises the following steps: 1) Subculture the ExpiCHO cells one day before the transient transfection, adjust the cell density to 2×10 ⁶ cells/ml, and place a shaker flask containing a certain volume of cells in a shaker for culture; 2) On the day of transient transfection, count the ExpiCHO cells, dilute with fresh medium to adjust the cell density to 4×10 ⁶ cells/ml, and the cell viability is above 95%, to obtain an ExpiCHO cell suspension; 3) For preparation before transfection, mix the prepared liquid A and liquid B and let it stand for a period of time to obtain a mixed drop; 4) Add the mixed drop into the ExpiCHO cell suspension, and cultivate according to the cell culture conditions. 3. the described method for enhancing ExpiCHO cell transient transfection antibody expression as claimed in claim 2, is characterized in that: described A liquid comprises the plasmid that contains antibody light and heavy chain and PBS damping fluid, the described plasmid that contains antibody light and heavy chain The ratio of antibody light chain-containing plasmid to antibody heavy chain plasmid is 1:1, and then the antibody light and heavy chain-containing plasmid is mixed with 600 μl of PBS buffer to obtain liquid A; the liquid B contains PEI transfection reagent and PBS buffer; the ratio of the total plasmid quantity of the antibody light and heavy chain plasmid to the cell number of the ExpiCHO cell suspension is 20 μg: 4×10 ⁷ cells-12×10 ⁷ cells; the antibody light and heavy chain The mass ratio of the total plasmid amount of the plasmid to the added amount of the transfection reagent is 1:2-1:6. 4. the method for the described enhanced ExpiCHO cell transient transfection antibody expression as claimed in claim 2, is characterized in that: described ExpiCHO cell will be subcultured once within 24 hours of transfection; 250ml～1000ml round-bottom shake flasks, use 125ml round-bottom shake flasks during transfection; the certain volume in step 1) is one of 10ml, 20ml, 30ml; The method is that the room temperature is not lower than 29°C, and the standing time is 10 to 20 minutes, or one of the standing water baths at 29°C; the cell subculture and cell culture conditions are 37°C, 8 % CO ² , shaker speed 110 rpm. 5. the method for the described enhanced ExpiCHO cell transient transfection antibody expression as claimed in claim 2, is characterized in that: the total plasmid quantity of described antibody light and heavy chain and the cell number ratio of described ExpiCHO cell suspension are 20 μ g : 8×10 ⁷ cells; the mass ratio of the total plasmid amount of antibody light and heavy chain plasmids to the added amount of the transfection reagent is 1:4. 6. the method for enhancing ExpiCHO cell transient transfection antibody expression as claimed in claim 2, is characterized in that: described ExpiCHO cell suspension also comprises serum-free medium, and described serum-free medium is Transpro CD01, and described ExpiCHO cell The suspension includes 4mmol/L-6mmol/L glutamine added before transfection. 7. the method for enhancing ExpiCHO cell transient transfection antibody expression as claimed in claim 1, is characterized in that, the method for promoting the antibody expression after the transient transfection of ExpiCHO cell comprises the steps: Recombinant ExpiCHO cells were obtained after transient transfection, and the temperature was lowered for 4-10 hours after transfection. Hydrolyzate was added 12-24 hours after transfection. The amount of hydrolyzate added was 3g/L-8g/L to promote transfection Cellular protein expression. 8. The method for enhancing ExpiCHO cell transient transfection antibody expression as claimed in claim 7, is characterized in that: the hydrolyzate is a plant protein hydrolyzate, and the plant protein hydrolyzate comprises Bacto malt powder, BBL plant protein hydrolyzate, Difco selected soybean peptone, Difco ultra-filtered yeast hydrolyzate, BBL yeast extract, Bacto yeast extract, Bacto yeast extract, Oxoid No. 1 plant peptone, Oxoid non-GMO soybean peptone, soybean SE50MK-NK, soybean SE50MK-NK, A mixture of one or more of soybean WGE80M-UF ultrafiltration type, Kerry Hypep-1510, Kerry Hypep-4601N, Kerry Hypep-YE and Oxoid No. 1 plant peptone. 9. The method for enhancing the expression of ExpiCHO cell transient transfection antibody as claimed in claim 7, characterized in that: at 18 hours after the transfection, the plant protein hydrolyzate of 3g/L～8g/L was added; The cooling culture conditions are 32°C, 8% CO2, and the shaking table speed is 110rpm; the plant protein hydrolyzate is a mixture of Bacto yeast extract, Kerry Hypep-1510 and Difco ultra-filtered yeast hydrolyzate, wherein Bacto yeast extract 5g/L, Kerry Hypep-15103g/L, Difco ultra-filtered yeast hydrolyzate 6g/L. 10. The application of the transient transfection method of ExpiCHO cells as claimed in claim 2 in recombinant protein expression.

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